I want to effect of metals on Superoxide Dismutase activity. Many protocols have suggested the use of riboflavin. However, I am not exactly clear in its role. Alternatively can anyone suggest me a protocol for SOD assay? The following are the two protocols I was referring to.
Hello,
SOD inhibits oxidation of a substrate with superoxide anion. Therefore, you should have SOD, substrate and superoxide anion in reaction medium. In the protocol you mention above, riboflavin are reduced by light and generates superoxide anion. This anion can change NBT to formazan, but SOD inhibits this reaction.
Other methods for SOD assay are oxidation of cytochrome C and pyrogallol. You can find the protocols in the web. However you should check which protocol is reliable fn your case.
I agree that riboflavin generates superoxide anion radicals in presence of methionine on exposure to light. SOD uses superoxide anion radicals as substrate. At the same time superoxide anion radicals have good pential to reduce tetrazolium to formazan.
In absence of SOD, superoxide anion radicals can reduce higher levels of tetrazolium to farmazan. If SOD activity is there, obviously suoeroxide anions radicals will be dismutated and hence will not be available for reduction of tetrazolium, Therefore, higher the SOD activity lesser would be the formation of formazan (i.e. reduction of tetrazolium).
Hi all,
i want to know which type of riboflavin can be used for SOD.
because chemical delivers ask which type of riboflavin is required.
riboflavin for biochemistry or vitamin B2?
please suggest
Hello,
I am doing some SOD assay with NBT and riboflavin, but in every protocole they add TEMED (tetramethylethylenediamine ). Does someone understand its role ?
Thank you
TEMED actually functions as a free radical stabilizer and also act as a catalyst in acrylamide gel preparation (PAGE) together with ammonium persulfate (APS). Briefly, the stabilization of the free radicals promote and improve acrylamide polymerization. Therefore, both APS and TEMED are two key reagents and without them, gels will not polymerize.
For SOD assay, consider reading these;
1- https://www.ncbi.nlm.nih.gov/pmc/articles/PMC542387/pdf/plntphys00136-0196.pdf
2- http://www.jbc.org/content/244/22/6049.full.pdf
3- http://www.sciencedirect.com/science/article/pii/0003269771903708
Hope this will help.
I have been doing SOD assay using riboflavin, NBT, Methionine in K-phosphate buffer pH 7.6 and accidentally I forgot to add EDTA in the reaction mixture, as a result the blue colour generation in the control mix (all components EXCEPT the enzyme extract) under light did not develop blue colour but very slowly and to very low level. when I repeated it and added EDTA I found the blue colour of the control mix (without enzyme extract) to develop fast as usual. Can anybody tell the role of EDTA in the oxidation of NBT and/or generation of superoxide from riboflavin? NOTE: The question here is not the effect of EDTA on SOD activity since control mix has NBT, riboflavin, methionine in pot. phosphate buffer pH 7.6
Hello, and
Can methionine be replaced? why compound ?. Because I want to measure sod activity and I don't have methionine. Thanks!
Andrea Vasconsuelo
Consider using this protocol.
https://www.sigmaaldrich.com/technical-documents/protocols/biology/enzymatic-assay-of-superoxide-dismutase.html
Best Regards
Is there something I can use instead of riboflavin? i've seen folic acid and glutamic acid but i don't have folic acid either
Is it necessary to incubate the same set of reactions in dark and subtract the OD of the dark incubated sample from the Light incubated sample?
Kindly check this out - riboflavin is readily able to donate an electron to spent SOD. Regards
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